This application will investigate the potential pathogenic properties of Treponema denticola utilizing biochemical and molecular genetic approaches. Specifically, the role of proteases and a fibronectin-binding adhesin elaborated by these organisms in attachment and destruction of oral cells and invasion of basement membranes will be examined. These spirochete properties could play a role in the development of periodontal diseases. The gene coding for the T. denticola protease exhibiting type IV collagenase activity has been recently isolated in this laboratory. Further characterization of the gene will be accomplished following nucleotide sequencing. T. denticola clone banks generated in E. coli will also be screened for other protease genes. Spirochete mutants defective in individual proteases will then be sought following insertional inactivation of the genes, and introduction into T. denticola by electroporation or by conventional mutagenesis. These mutants will be utilized to examine the role of the enzymes in model cellular attachment and invasion systems. Likewise, the gene coding for the fibronectin-binding adhesin (FBA) will be isolated in lambda gtll clone banks. The protein product will be purified and utilized to produce monospecific antibody to localize the adhesin in T. denticola. The gene will be sequenced and the deduced amino acid sequence utilized to identify functional domains of the protein following site-directed mutagenesis. In addition, the gene will be used to generate FBA-negative mutants of the spirochete for assessment of the role of the gene product in attachment to basement membranes. These approaches should provide new information regarding the pathogenic potential of T. denticola and its possible role in periodontal diseases.